Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes that are present in very low concentration in biological fluids. Such analytes include, for example, antigens, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
The analyte, which is the target of the assay, is referred to herein as the ligand. Compounds which specifically recognize the ligand and react to form complexes with the ligand are referred to herein as receptors. The receptor and the ligand form a conjugate pair. Any member of the pair can function as a receptor or a ligand.
In the case of a competitive assay, a labeled analyte, including labeled immuno-competent derivatives and analogs of such analyte, are typical components of the assay; whereas, in the case of a sandwich assay, a labeled receptor for the analyte is typically employed. These are referred to herein as the labeled ligand and labeled receptor, respectively.
In competitive binding immunoassays, a labeled ligand is placed in competition with unlabeled ligand for reaction with a fixed amount of the appropriate receptor. Unknown concentrations of the ligand can be determined from the measured signal of either the bound or unbound (i.e. free) labeled ligand. The reaction proceeds as follows: EQU ligand+labeled ligand+receptor-substrate&lt;=&gt;ligand-receptor-substrate+labeled ligand-recep tor-substrate.
In an alternative immunoassay format known as a sandwich immunoassay or immunometric assay, the ligand is contacted with two or more receptor molecules which bind to the ligand at different epitopic sites. One receptor is typically appropriately labeled and the other is either immobilized on a solid substrate or is capable of being immobilized thereon. The amount of ligand is directly proportional to the amount of bound complex among the ligand and the two receptors. This is illustrated as follows: EQU substrate-receptor.sub.1 +ligand+receptor.sub.2 -label&lt;=&gt;substrate-receptor.sub.1 -ligand-receptor.sub.2 -label
Conventional labels include radioactive tags, enzymes, chromophores, fluorophores, stable free radicals and enzyme cofactors, inhibitors and allosteric effectors.
Immunoassay analytical elements are known from U.S. Pat. Nos. 4,517,288 and 4,258,001. In general, such elements comprise receptors, such as antibodies for a ligand, immobilized in a particulate layer. In addition the element usually contains a reagent system that, through interaction with a bound or unbound species, results in a signal that can be correlated to the concentration of ligand in a sample. In use, the sample may be combined with an enzyme labeled ligand and applied to the element. After a time, a solution containing a substrate for the labeled ligand is applied to the particulate layer. The reaction with the substrate is catalyzed by the enzyme label to form a reaction product that ultimately causes a signal, for example, color to develop. The reflection density of the color can be correlated to the concentration of the ligand in the sample. Similar signal development systems are known for other known conventional labels such as radioactive tags, chromophores, fluorophores, stable free radicals, and enzyme cofactors, inhibitors and allosteric effectors.
Multilayer immunoassay elements are dry-film elements which use the above described immunoassay principles to measure analytes in fluid samples. In competitive assay elements, the rate of color formation (or other signal) is inversely correlated to the amount of analyte present and in sandwich assay elements, the rate of color formation (or other signal) is directly correlated to the amount of analyte present. Also, the rate of color formation (or other signal) is directly proportional to the activity of the enzyme labeled analyte, e.g. drug, or enzyme labeled receptor bound to the immobilized receptor. For the immunoassays to maintain a stable calibration, none of the enzyme activity (measured rate) can be lost in any of the slides during the specified calibration period.
Frequently, immunoassay elements are supplied to customers in plastic "cartridges" containing 50 separate elements from which one element may be removed at a time as needed. The elements are stacked one on top of another so that the lower 49 elements in the cartridge all have their top surfaces covered by the element above. However, the top element in the stack has no such covering and therefore the surface of that element is exposed to environmental factors to which the other 49 elements are not. For example, the top (or first) element is more exposed to air flow and light than the remainder of the elements when the cartridges are being handled during manufacturing or when the cartridges are in the element supplies of the clinical analyzers.
During storage, prior to use, the cartridges themselves are stored in sealed, foil-lined bags. However, the top element is still more exposed to the residual air and humidity inside the sealed bags than the other 49 elements.
It has been found that, when a common test fluid was reacted with the elements in a cartridge, the rate of color formation observed in the top (or first) element was always lower than the rate of color formation observed when the same test fluid was applied to elements below the top element in the same cartridge. This is referred to as first slide bias.